Tyrosine kinase serves as a growth factor receptor or intracellular signal transducer and can be aberrantly activated through a variety of mechanisms (70
). In hematopoietic cells, a common mechanism for activation of tyrosine kinase is the occurrence of fusion products as a consequence of balanced translocations (51
). Similarly, activated and overexpressed tyrosine kinases have been reported in association with solid cancers, especially FGFRs and their ligands, which play a major role in cancers of the stomach, breast, thyroid, prostate, and pancreas. We recently found FGFR1 amplified and overexpressed in breast cancers (77
FOP-FGFR1 is the fusion product of the t(6;8) translocation associated with a stem cell leukemia-lymphoma syndrome in which both lymphoblastic lymphoma and acute myelogenous leukemia develop in patients (65
). Understanding the mechanisms leading to this syndrome could provide information on the biology of the hematopoietic stem cell itself. In this study we have shown that the FGFR1 aberrant tyrosine kinase is constitutively phosphorylated and transforms Ba/F3 cells to IL-3-independent cell survival via an antiapoptotic effect. Mutation of the ATP binding site in the catalytic domain of the fusion protein abrogates activation and cell survival. Cell survival induced by FOP-FGFR1 is also abrogated by mutation of the PLC-γ-binding site. Experiments described here also show that the constitutively activated FOP-FGFR1 tyrosine kinase utilizes cooperation between effector proteins, including STAT1 and STAT3, PLC-γ, MAPK, PI3K, and mTOR/p70S6K
, to facilitate signal transduction and promote cell survival.
In addition to the roles of STAT proteins in normal cell signaling, recent studies have demonstrated that diverse oncoproteins can activate specific STATs and that constitutively activated STAT signaling directly contributes to oncogenesis (7
). While STAT activation is a common characteristic of leukemias, the specific patterns of activated STATs and the manner by which STAT activation occurs vary with each disease (49
). FOP-FGFR1 stimulates phosphorylation and activation of STAT1 and STAT3. Recently, Hart et al. (36
) showed that FGFR derivatives which contain the same deletion of the extracellular domain of FGFR1 as FOP-FGFR1 were capable of inducing morphological transformation and differentiation of PC12 cells and stimulating phosphorylation and activation of STAT1 and STAT3. Constitutive activation of STAT1 and STAT3 are also found in Epstein-Barr virus-related lymphoma cells (81
), in Burkitt lymphomas, and in multiple myeloma (8
). STAT3 activation plays an important role in preventing apoptosis (26
). Ectopic expression of FGFR3 as a result of the t(4;14) translocation promotes myeloma cell proliferation and prevents apoptosis via increased phosphorylation of STAT1 and STAT3 and higher levels of BCLXI
). These observations are relevant given the fact that STAT1 and STAT3 are constitutively activated by FOP-FGFR1, which induces an antiapoptotic effect. In striking contrast to a broad spectrum of tyrosine kinase fusions associated with hematological malignancy, including BCR-ABL, TEL-JAK2, and TEL-PDGFRβ, we found that FOP-FGFR1 did not activate STAT5. Thus, FOP-FGFR1 represents the second evidence of nonactivation of STAT5 by a tyrosine kinase fusion protein. Indeed, a recent study showed that TEL-TRKC-mediated transformation of Ba/F3 and development of myeloproliferative disorder do not require activation of STAT5 (50
). Most significantly, STAT5 activation does not play a necessary role in spite of its activation in the disease induction caused by BCR-ABL or v-ABL (48
). The significance of activation of specific STAT awaits the elucidation of target genes specific to FOP-FGFR1-mediated survival. In particular STAT3, whose activation is controlled by mTOR (71
), is persistently activated in many human cancers and causes cellular transformation (9
Our results establish that the MAPK pathway is critical for the transformation of the bone marrow-derived Ba/F3 cell line by FOP-FGFR1. In normal and transfected cells, FGF-mediated FGFR activation results in a strong activation of the MAPK cascade by means of the recruitment of the GRB2-SOS complex to the plasma membrane via phosphorylation of the docking proteins SHC and FRS2/SNT (44
). The specific sites for FRS2 binding within the FGFR juxtamembrane portion are deleted in the fusion protein FOP-FGFR1, and we have shown that FOP-FGFR1 is associated with SHC that is tyrosine phosphorylated. This resulted in reduced MAPK phosphorylation. As a consequence, FOP-FGFR1 is a less potent activator of MAPK than FGFR1. In addition, we showed that the MEK specific inhibitors induced complete inhibition of MAP kinase activation as well as cell survival triggered by FOP-FGFR1 in the absence of ligand, contrary to the wild-type FGFR1 for which an incomplete inhibition was found in response to FGF1 stimulation (this work and reference 58
). In agreement with other studies (52
) on the FGFR1 autophosphorylation roles in signaling, we have shown that tyrosine 511 of FOP-FGFR1, the binding site for the SH2 domain of PLC-γ (Y766 in the wild-type FGFR1), is not required for the activation of MAPK pathway.
In addition to the MAPK cascade, the PI3K pathway must also be active for FOP-FGFR1 to exert its survival effect. The involvement of PI3K in cell transformation has been demonstrated in several cell lines (2
). PI3K plays an important role in regulation of cell proliferation and apoptosis. In addition, constitutive PI3K activity has been observed in cancerous cells and, therefore, may contribute to the malignant transformation of cells. PI3K is involved in signal transduction downstream of most tyrosine kinases (38
). The FGFRs lack optimal binding motifs for the PI3K and FGF-induced PI3K activity is difficult to detect in vitro (43
). PI3K is constitutively activated through an unknown mechanism in FOP-FGFR1-expressing Ba/F3 cells. LY294002 and wortmannin completely abolished the survival of FOP-FGFR1-expressing Ba/F3 cells. Moreover, recent studies showed that cytokine receptors lacking direct PI3 kinase binding sites activate AKT via PI3K pathway, thereby regulating cell survival/or proliferation (32
). Several studies have recently established a link between the PI3K/AKT pathway and human cancers via defects in PTEN (reviewed in reference 13
). In addition, SHC, which is recruited by the fusion protein, might be required for PI3K activation in FOP-FGFR1 as well.
PI3K, AKT, and their downstream effectors mTOR and p70S6K
have recently emerged as components of a major signaling pathway that is dedicated to cell growth and survival via protein translation (6
). mTOR appears to be an obligatory mediator of the oncogenic signal issued by PI3K or AKT (3
). Although the complete target spectrum of mTOR remains to be determined, it is clear that mTOR functions as an important regulator of translation (3
). For the first time the data presented here point to a deregulated PI3K/AKT and mTOR/p70S6K
signaling induced by an aberrant tyrosine kinase fusion protein.
Mutation in the binding site of PLC-γ of FOP-FGFR1 (Y511 equivalent of Y766 in the FGFR1 sequence) abrogates the phosphorylation of PLC-γ, PI3K, and p70S6K
. This observation shows the importance of this autophosphorylating site, which may constitute a multidocking site for effector proteins in FOP-FGFR1 mediated cell survival. The importance of the multifunctional docking site of the MET receptor tyrosine kinase in mediating several cellular responses has been demonstrated (29
). Signaling via growth factor frequently results in the concomitant activation of PLC-γ and PI3K. A cross talk scenario between PLC-γ and PI3K was described recently to activate growth factor receptors following stimulation (27
), thus revealing intricated signaling pathways. It has been suggested that PI3K may act as an intermediate in the activation of the MAPK cascade in erythroid progenitors (41
). Finally, the ability of different kinase cascades to independently protect hematopoietic cells from apoptosis was recently demonstrated (61
). With regards to antiapoptotic signaling via the IL-3 receptor, both PI3K and activation of the MAP signaling pathway appear to be important in Ba/F3 cells (16
). Our results strongly suggest that FOP-FGFR1 activates the same signaling pathways as IL-3 in generating antiapoptotic signals.
In hematopoietic cells, multiple receptors are able to prevent apoptosis via the activation of PI3K. AKT is a downstream component of the PI3K pathway, and its phosphorylation is tightly associated with its activation (10
). We showed that activation of the antiapoptotic mediator AKT by FOP-FGFR1 was abrogated by wortmannin and LY294002, indicating that FOP-FGFR1 activates AKT in a PI3K-dependent manner. Recently, AKT was found to phosphorylate procaspase-9 and to inhibit its protease activity, thus promoting cell survival (15
). Our results show that caspase-9 is inactivated in FOP-FGFR1-expressing cells, strongly suggesting that the antiapoptotic effect elicited by FOP-FGFR1 is related to the caspase cascade. In addition, the analysis of a more general indicator of cell viability, i.e., BCL2, showed higher expression in FOP-FGFR1-expressing cells than in other transfectants. Suppression of apoptosis induced by growth factor withdrawal was recently reported by an oncogenic form of c-CBL by a mechanism that involves the overexpression of BCL2 (34
). Overexpression of BCL2 has also been associated with late myelodysplastic syndrome types or progression to overt leukemia (21
). It will be interesting to see whether the BCL2 overexpression response is under the control of the PI3K/mTOR pathway.
In conclusion, FOP-FGFR1 main activity is the promotion of cell survival through connection with intricated signaling pathways (Fig. ). Importantly, our results with FOP-FGFR1 establish that constitutive activation of the MAPK and PI3K pathways can contribute to the neoplastic state. The characterization of the effects of FOP-FGFR1 on murine hematopoietic stem cells should illuminate the critical signaling pathways and should orientate the development of drugs for the myeloproliferative disorder treatment.
FIG. 13 Schematic representation of signal transduction molecules interacting with FOP-FGFR1 compared to wild-type FGFR1. Both PLC-γ and MAPK are recruited to activated FGFR1 following FGF stimulation. In addition, whereas Pl3K was not detected, AKT was (more ...)